Kinetic Spectrophotometric Method For The Determination of Ramipril

AAPS PharmSciTech 2005; 6 (3) Article 68 (http://www.aapspharmscitech.org).
Kinetic Spectrophotometric Method for the Determination of Ramipril

in Pharmaceutical Formulations
Submitted: February 1, 2005; Accepted: May 24, 2005; Published: October 27, 2005
Nafisur Rahman,1 Yasmin Ahmad,1 and Syed Najmul Hejaz Azmi1

1
Department of Chemistry, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India
ABSTRACT
The objective of this research was to develop a kinetic spectrophotometric method for determination of ramipril in
pure form and pharmaceutical formulations. The method
was based on the reaction of carboxylic acid group of the
drug with a mixture of potassium iodate (KIO3) and potassium iodide (KI) in aqueous medium at room temperature. The reaction is followed spectrophotometrically by
measuring the increase in absorbance at 352 nm as a function of time. The initial-rate and fixed-time methods were
adopted for constructing the calibration curves. Both the
calibration curves were linear in the concentration range of
10.070.0 g mL-1. The detection limits were 0.02g mL-1
and 0.15-g mL-1 for initial rate and fixed time methods,
respectively. The proposed methods are validated statistically and through recovery studies. The point and interval
hypothesis tests have been performed confirming that there
is no significant difference between the proposed methods
and the reference method. The experimental true bias of
all samples is less than 2%. The methods have been
successfully applied to the determination of ramipril in
tablets and capsules.
KEYWORDS: initial-rate method, fixed-time method,

ramipril, pharmaceutical formulations, spectrophotometry,
validationR
INTRODUCTION
Ramipril, 2-[N- [(S)-1-ethoxy carbonyl-3-phenyl propyl]-Lalanyl]-(1S, 3S, 5S)-2-azabicyclo [3,3,0]-octane-3-carboxylic
acid [CAS: 87333195] is a prodrug1 which is rapidly
hydrolyzed with the cleavage of an ester group through
hepatic metabolism forming an active metabolite ie, ramiprilat. This prodrug itself is a poor inhibitor of angiotensin
converting enzyme (ACE) but its active metabolite has a
higher affinity for ACE, thus blocking the conversion of the
angiotensin I to the angiotensin II, a highly potent vaso-
Corresponding Author: Nafisur Rahman, Department of

Chemistry, Aligarh Muslim University, Aligarh-202002,
Uttar Pradesh, India. Tel: +91-571-2703515.
E-mail: cht17nr@yahoo.co.in
constrictor and there by leading to a reduction in vasopressor

activity and a decrease in peripheral vascular resistance.2,3
The drug is officially listed in British Pharmacopoeia,4 which
describes a potentiometric titration procedure for its assay
in bulk and dosage form.
The estimation of ramipril along with hydrochlorothiazide
in binary mixture was performed by derivative compensation
technique5 as well as zero crossing derivative technique.6,7
Two spectrophotometric methods have been reported for the
assay of drug in commercial dosage forms, which are based
on the formation of ternary complex of the drug with Cu (II)
and eosin8 and Fe (III) and ammonium thiocyanate.9 The
drug content in pharmaceutical formulations has been determined spectrophotometrically in visible region based on
the charge transfer reaction of ramipril with -acceptors
such as 7,7,8,8,tetracyanoquinodimethane and p-chloranilic
acid and subsequently measuring the absorbance at 840 and
520 nm, respectively.10 Two extractive spectrophotometric
methods11 have been recommended based on extractive
ion-pair complex of the drug with picric acid and bromocresol green. Potassium permanganate oxidizes ramipril in
alkaline medium resulting in the formation of bluish green
colored complex peaking at 610 nm.12 The quantitation of
ramipril13 has been done by spectrophotometry and fluorimetry utilizing the reaction of the drug with 7-fluoro-4nitrobenzo-2-oxo-1,3-diazole which exhibits maximum
absorbance at 460 nm, and maximum fluorescence intensity at 530 nm after excitation at 465 nm. The literature is
still poor in analytical procedures based on kinetic spectrophotometry for the determination of drug in pharmaceutical formulations. Therefore, there is a need for a simple
kinetic spectrophotometric method for the determination of
ramipril in commercial dosage forms.
This paper describes the development and validation of a
kinetic spectrophotometric method for the determination of
ramipril in pharmaceutical formulations. The method is
based on the reaction of COOH group of ramipril with a
mixture of iodide-iodate in aqueous medium resulting in
the formation of yellow color, which absorbs maximally at
352 nm. The absorbance increases with time and therefore,
two calibration procedures ie, initial-rate and fixed time
methods are adopted for the determination of ramipril in
commercial dosage forms.
E543
MATERIALS AND METHODS
Fixed-Time Method
Apparatus
In the fixed time method, the absorbance at 352 nm of each

sample solution was measured at a preselected fixed time
against a reagent blank prepared similarly. The calibration
curve was constructed by plotting the absorbance against
the final concentration of the drug. The amount of the drug
in each sample was computed either from calibration curve
or regression equation.
A Shimadzu UV-visible 1601 spectrophotometer (Model

no1601, Kyoto, Tokyo, Japan) with matched quartz cells
was used for all spectral and absorbance measurements.
A water bath shaker (NSW 133, New Delhi, India) was used
to control the heating temperature for the formation of degraded ramipril.
Standard and Reagents
All chemicals and reagents used were of analytical or pharmaceutical grade.
Ramipril reference standard (Batch No. TR001FO2, purity
99.58%) was kindly provided by Dr. Reddys Laboratories
Limited (Andhra Pradesh, India). Standard solution of ramipril (0.5 g mL-1) was prepared by dissolving 50 mg in
100 mL distilled water. This solution was used to prepare
calibration curve and quality control samples. Quality control
samples were prepared at three concentration levels of 20,
40 and 70 g mL-1. The solution is stable for at least 1 week
if kept stored in a cool and dark place.14 Pharmaceutical
formulations of ramipril such as Hopace-1.25 (Cardicare,
Bangalore, India), Ramipres-1.25 (Cipla, Mumbai, India),
Ramace-1.25 (AstraZeneca, Mumbai, India) and Variace1.25 (Win Medicare, New Delhi, India) were obtained from
commercial sources.
A 0.15 M KI (Fluka Chemie AG, Switzerland) solution
was freshly prepared in distilled water. The solution was
standardized by the recommended procedure.15 A 0.1 M
KIO3 (Fluka Chemie AG, Switzerland) was also freshly
prepared in distilled water.
Recommended Procedures for the
Determination of Ramipril
Initial Rate Method
Aliquots of 0.21.4 mL reference standard solution of
ramipril (0.5 mg mL-1) were pipetted into a series of 10 mL
standard volumetric flasks. In each flask, 2.2 mL of 0.1 M
KIO3 followed by 3.3 mL of 0.15 M KI were added and
then diluted to volume with distilled water at 25 1C. The
contents of each flask were mixed well and the increase in
absorbance at 352 nm was recorded as a function of time
against the reagent blank prepared similarly. The initial
rate of the reaction () at different concentrations was obtained from the slope of the tangent to the absorbance-time
curve. The calibration curve was constructed by plotting the
logarithm of the initial rate of reaction (log ) vs the
logarithm of the molar concentration of the ramipril (log C).
The amount of the drug was obtained either from the calibration graph or the regression equation.
Procedure for the Determination of Ramipril in

Pharmaceutical Formulations
The sample solution containing ramipril at a concentration
of 0.5 mg mL-1 was prepared. The powdered contents of
10 capsules of 1.25 mg (or 5.0 mg) strength were obtained
by gentle tapping and the hard gelation shells were discarded. The ramipril was extracted into 510 mL portions
of methanol by shaking. Similarly, 10 tablets of 1.25 mg
(or 5.0 mg) strength were taken in 10 mL methanol and
left for 10-minute for complete dispersion and then extracted into 510 mL portions of methanol by shaking and
filtered through Whatmann No. 42 filter paper. The residue
was washed well with methanol for complete recovery of
the drug. The methanol was evaporated to dryness under
vacuum and the remaining drug was dissolved in an appropriate volume of distilled water to give a concentration of 0.5mg mL-1. The assay was completed following
the recommended procedures for the determination of
ramipril.
Procedure for Reference Method8

Aliquots of 0.21.0 mL of 0.1% ramipril were pipetted into
a series of 50.0 mL separating funnels. Into each separating
funnel, volume of solution was adjusted to 10.0 mL with
distilled water and 3.0 mL of 0.2% Cu (II) sulfate solution
followed by 1.0 mL of 0.1% eosin solution were added
successively and mixed well. The funnels were shaken
vigorously with 3.03.0 mL portions of chloroform for 1.0minute, and then allowed to pass the organic layer through
anhydrous sodium sulfate into a 10.0 mL standard
volumetric flask. The volume of chloroform layer was
made up to 10.0 mL and the absorbance was measured at
535 nm against the reagent blank prepared similarly. The
amount of the drug in a given sample was computed either
from calibration graph or regression equation.
Validation
The proposed method has been validated for specificity,
linearity, precision, accuracy, and recovery.
E544
Specificity
Samples of composite of ramipril capsules (1.25 mg) were
subjected to stress conditions of light, heat, acid, base and
oxidants. All stressed samples were analyzed for ramipril
content and compared with an unstressed time zero reference solution. The time zero solution provided a reference assay value for the unstressed product. The content of
degradation in the stressed and control samples was calculated relative to this assay value.
Linearity
For evaluation of linearity, determination of ramipril was
done at seven concentration levels: 10.0, 20.0, 30.0, 40.0,
50.0, 60.0 and 70.0 g mL-1. Each concentration was
analyzed for five times.
Precision and Accuracy
Three concentrations within the linearity range were selected: 20.0, 40.0, and 70.0 g mL-1. Five sample solutions
of each concentration were prepared and analyzed within
one day. This assay was too repeated for five consecutive
days. The intra and inter precision and accuracy were also
determined by analyzing the quality control samples that
were tested five times in one day and on five consecutive
days.
thus showing higher sensitivity at 352 nm. Therefore, the

absorbance measurements for the determination of ramipril were made at 352 nm. The equilibrium is attained in
~50-minute. Therefore, a kinetically based spectrophotometric method was developed for the quantitative determination of ramipril by measuring the increase in absorbance at
352 nm as a function of time.
Mechanism of the Color Reaction
It has been suggested that water-soluble acidic compounds
liberate iodine from a solution containing both KIO3 and
KI according to the reaction16:
5I IO3 6H 3H2 O 3I2
A yellowing of the solution reveals the occurrence of the
reaction. The yellow color of the solution is due to the
formation of I2, which immediately converted into triiodide
ions in the presence of iodide ions (I2 I I3 ) exhibiting
absorption maxima at 290 nm and 360 nm.17 Ramipril, a
water-soluble ACE inhibitor, possesses COOH group in
its moiety and hence undergoes a similar reaction with
iodide-iodate mixture resulting in the evolution of iodine.
The liberated iodine immediately reacts with potassium
iodide to give triiodide ions showing absorption maxima at
298 nm and 352 nm. The reaction sequence is shown in
Recovery Studies
To study the accuracy of the proposed method and to check
the interference from excipients used in the formulations,
recovery experiments were performed by standard addition
method. For this, 4.0 mL (or 6.0 mL) of sample solution
(0.5 mg mL-1) was transferred into a 100.0 mL volumetric
flask followed by 4.0 mL (or 8.0 mL) of reference standard
solution (0.5 mg mL-1) and volume was completed to the
mark with distilled water. The nominal value was determined by the proposed procedures.
RESULTS AND DISCUSSION

Spectral Studies
The spectrum of reference pure drug of ramipril in aqueous
solution shows two absorption bands at 210 nm and 234 nm
(Figure 1 A). The addition of aqueous solutions of KI and
KIO3 to the drug solution causes change in the absorption
spectrum with new characteristic bands peaking at 298 and
352 nm (Figure 1 C). The reagent blank solution containing
KI and KIO3 shows one peak at 275 nm and a negligible
absorbance (0.06) at 352 nm when measured against distilled water as a reference (Figure 1B). The absorbance obtained at 352 nm is higher than the absorbance at 298 nm,
Figure 1. Absorption spectra of (A) 1.0 mL standard ramipril

solution (0.05%) in distilled water, (B) blank solution: 3.3 mL of
0.15 M potassium iodide, 2.2 mL of 0.1 M potassium iodate,
(C) sample solution: standard ramipril (50.0 g mL-1) + 3.3 mL
of 0.15 M potassium iodide and 2.2 mL of 0.1 M potassium
iodate. Each set is diluted to 10 mL with distilled water.
E545
Figure 2. The confirmatory test for the presence of iodine

in the final solution of the drug is established by the blue
color, which appears on addition of starch solution.18
Optimization of Variables
Preliminary experiments were performed to determine the
optimum conditions of the variables used in the estimation
of ramipril. The influence of the variables on the rate of
reaction was studied and optimized. The optimum values of
the variables were maintained throughout the experiment.
Effect of the Concentration of Potassium Iodate
The effect of the concentration of potassium iodate was
studied by treating 50.0 g mL-1 ramipril with 3.3 mL of
0.15 M KI and varying volumes (0.52.5 mL) of 0.10 M
KIO3. The kinetic slope (tan =dA/dt) of the absorbancetime curves obtained at different volumes of KIO3 showed
that the initial rate of reaction was increased with increasing volume of KIO3 and became constant at 2.0 mL; above
this volume, the initial rate of reaction remained unchanged
(Figure 3A). Therefore, 2.2 mL of KIO3 (0.10 M) was
used in all measurements.
Figure 3. Effect of the volume of (A) 0.10 M potassium iodate,

(B) 0.15 M potassium iodide on the initial rate of reaction.
beyond this volume, the initial rate remained constant. Therefore 3.3 mL of 0.15 M KI was recommended for determination procedures.
Effect of the Concentration of Potassium Iodide

The influence of the volume of 0.15 M KI on the rate of
reaction was investigated in the range of 0.53.5 mL. The
initial rate of reaction (Figure 3B) was increased with
increasing volume of KI and became constant at 3.0 mL;
Analytical Data
Under the optimized experimental conditions, the assay of
ramipril was performed in presence of excess concentration
of KIO3 and KI in aqueous solutions with respect to ramipril
concentration. Therefore, a pseudo zero order reaction condition was worked out with respect to the concentration of
the reagents. The kinetic plots (Figure 4) are all sigmoid in
nature and therefore, the initial rate of reaction was obtained by measuring the slopes (tan = dA/dt) of the initial
tangent to the absorbance-time curves at different concentrations of the drug. The order with respect to ramipril was
evaluated by plotting the logarithm of the initial rate of
reaction vs logarithm of the molar concentration of ramipril
and was found to be one.
The initial rate of reaction would follow a pseudo first
order and obeyed the following rate equation:
Rate
A
kC n
t
where k is the rate constant, C is the concentration of

ramipril, n is the order of reaction. The logarithmic form of
the equation is written as:
lograte logk nlogC
Figure 2. Reaction sequence of the proposed method.
E546
A calibration curve was constructed by plotting the logarithm of the initial rate of reaction (log ) vs logarithm of
initial concentration of ramipril (log C), which showed a
linear relationship over ramipril concentration of 10.0
70.0 g mL-1. The linear regression analysis using the
method of least square treatment of calibration data (n = 7)
was made to evaluate slope, intercept and correlation coefficient. The regression of log rate vs log C gave the following linear regression equation:
lograte 3:5665 1:0277logC
with a correlation coefficient (r) of 0.9999. The value of n

representing order of reaction in the regression equation is
one, confirming the first order reaction with respect to the
ramipril concentration. The limit of detection (LOD) for
initial rate method was calculated19 by statistical treatment
of calibration data (n = 7) by considering seven calibration
points using the following equation:

t 2 n2 1=2
LOD S0
b
n1
where n is the number of standard samples (n = 7), t is the

value of students t for n-2 degrees of freedom at 95%
confidence level, b is the slope of the regression line and
S02 is the variance characterizing the scatter of the experi-
Table 1. Spectral and Statistical Data for the Determination of

Ramipril by Initial Rate Method
Parameters
Initial Rate Method
max(nm)
Linear dynamic range (g ml-1)
Regression equation
352
10.070.0
log(rate) = 3.5665 +
1.0277 log C
6.388 103
3.5665
3.595 102
9.243 102
1.0277
8.768 103
2.254 102
0.9998
4.080 105
0.020
S*0
Intercept (a)
Sa
tSa
Slope (b)
Sb
tSb
Correlation coefficient (r)
Variance (S02 )
Detection limit (g mL-1)
*Standard deviation of the calibration line.
Confidence interval of the intercept at 95% confidence level.
Confidence interval of the slope at 95% confidence level.
mental data points with respect to the line of regression.

The variance was calculated using the equation20:
S02 logexp logreg 2 =n2
Linear dynamic range, correlation coefficient, variance, detection limit, standard deviations and confidence limits for
slope and intercept of the calibration line are summarized
in Table 1. The low values of variance confirmed negligible
scattering of the experimental data points around the line
of regression and good sensitivity of the proposed method.
Fixed Time Method
Figure 4. Absorbance-time curves for the reaction of varying

concentration of ramipril with potassium iodate and potassium
iodide. Concentration of ramipril (g mL-1): (A) 10, (B) 20,
(C) 30, (D) 40, (E) 50, (F) 60, (G) 70.
In this method, the absorbance of a yellow colored solution

(max = 352 nm) was recorded at a preselected fixed time.
Calibration graphs of absorbance vs initial concentration of
ramipril at seven concentration levels were plotted at a
fixed time of 2, 4, 6, 8, and 10-minute. Beers law limit,
molar absorptivity, linear regression equation, coefficient of
correlation, detection limit, variance, standard deviation
and confidence limits for slope and intercept are summarized in Table 2. Test of significance of the intercepts, a, of
regression lines of the fixed time method at different intervals of time showed that these values of a did not differ
significantly from the theoretical value, zero. For this, a
simplified method was used to calculate the quantities t
from the relation t = a/Sa21,22 and their comparison with
the tabulated data from the students t-distribution.23 The
t-values for fixed time method at 2, 4, 6, 8 and 10-minute
do not exceed the 95% criterion (t = 2.571). It confirmed
E547

Table 2. Spectral and Statistical Data for the Determination of Ramipril by Fixed Time Method
Fixed-time method
Parameters
Beers law limit
(g mL-1)
Molar absorptivity
(L mol cm-1)
Linear Regression
equation
Intercept
Sa
tSa
Slope
Sb
tSb
Correlation
coefficient (r)
Variance (So2)
S*o
Detection limit
(g mL-1)
t =a / Sa
2-minute
4-minute
6-minute
8-minute
10-minute
10.070.0
10.070.0
10.070.0
10.070.0
10.070.0
5.161103
5.954103
6.591103
7.288103
7.989103
A = 5.714104 +
1.238102 C
5.714104
9.389104
2.414103
1.238102
2.099105
5.398105
0.9999
A = 7.143104 +
1.428102 C
7.143104
1.264103
3.249103
1.428102
2.826105
7.264105
0.9999
A = -2.857104 +
1.583102 C
-2.857104
8.601104
2.211103
1.583102
2.082105
5.353105
0.9999
A = -2.571103 +
1.755102 C
-2.571103
1.846103
4.746103
1.755102
4.128105
1.061104
0.9999
A = 5.714104 +
1.916102 C
5.714104
1.284103
3.302103
1.916102
2.871105
7.382105
0.9999
1.234106
1.111103
0.211
2.235106
1.495103
0.246
1.036106
1.018103
0.151
4.771107
2.184103
0.292
2.309106
1.520103
0.186
0.609
0.565
0.332
1.393
0.445
*Standard deviation of the calibration line.
Calculated t-value, which is less than the theoretical value of t (2.365) at 95% confidence level.
that the calculated intercepts for the fixed time method are
not significantly different from zero. Thus, the fixed time
methods are free from constant errors independent of the
concentration of ramipril. It is apparent from Table 2 that
the values of intercept, standard deviation of the slope and
intercept and detection limit were found to be lowest at a
fixed time of 6-minute. Therefore, on the basis of lowest
values of these parameters, the fixed time of 6.0-minute
was recommended for the assay of ramipril in pharmaceutical formulations.
ramipril diketopiperazine (ester), which is a principal degradant of the ramipril. It was found that the specified
degradant did not react with either of the reagents.
Specificity
The samples were stressed by light, acid and oxidants such

as potassium persulfate, ammonium molybdate, sodium metavanadate, KBrO3, N-bromosuccinimide, chloramine T, and
sodium nitrate for 2, 5 and 5-day-time points, respectively.
The specificity of the proposed method was evaluated by

determining the ramipril concentration in the presence of
varying amounts of degraded product of ramipril such as
The various excipients commonly used in dosage forms

such as sodium stearyl fumarate, magnesium stearate, starch,
lactose, and talc did not interfere with the assay procedure.
The solubility of stearic acid in distilled water is negligible,
it is only soluble in chloroform, acetone and ether,24 and
therefore stearic acid is not interfering with the determination process as the developed method is performed only
in distilled water.
Table 3. Intra Day Assay: Test of Precision of the Proposed Methods for the Determination of Ramipril
Concentration (g mL-1)
Proposed Methods
Theoretical
Initial rate method
20.0
40.0
70.0
20.0
40.0
70.0
Fixed time method
Nominal SDa
20.012
39.994
69.988
20.031
39.993
70.037
Recovery RSD* (%)
0.113
0.105
0.117
0.053
0.053
0.072
*Mean for five determinations.
SAE, standard analytical error.
CL, confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).
E548
100.058
99.985
99.983
100.153
99.980
100.053
0.563
0.264
0.167
0.264
0.133
0.103
SAE
CL
0.050
0.047
0.052
0.024
0.024
0.032
0.140
0.131
0.145
0.066
0.066
0.089

Table 4. Inter Day Assay: Test of Precision of the Proposed Methods for the Determination of Ramipril
Concentration (g mL-1)
Proposed methods
Theoretical
Initial rate method
20.0
40.0
70.0
20.0
40.0
70.0
Fixed time method
Recovery RSD* (%)
Nominal SD*
19.992
40.105
70.044
20.005
40.031
69.987
0.124
0.253
0.182
0.056
0.085
0.072
99.957
100.262
100.062
100.026
100.077
99.981
0.618
0.631
0.260
0.282
0.213
0.103
SAE
CL
0.055
0.113
0.082
0.025
0.038
0.032
0.153
0.314
0.260
0.070
0.106
0.089
SAE, standard analytical error.
CL, confidence limit at 95% confidence level and four degrees of freedom (t = 2.776).
Accuracy and Precision of the Proposed Methods
It was observed that stress by such conditions did not cause

significant degradation. There was no change in the absorption spectra of the reference drug and stressed sample solutions. However, the samples degraded significantly when
stressed by base and heat for 1 and 2 hours, respectively. All
stressed samples (light, acid and oxidants) and unstressed
reference solution were analyzed for ramipril content, which
gave acceptable recoveries of the drug. Thus the proposed
method is stability-indicating assay for the determination of
intact ramipril in the presence of its degradation products.
The accuracy and precision of the proposed methods was

established by measuring the content of ramipril in pure
form at three different concentration levels (20.0, 40.0 and
70.0 g mL-1). The intra day precision of the proposed
methods was performed by carrying out five independent
analyses at each concentration level within one day (Table 3).
In the same manner, the inter day precision was also evaluated by measuring the ramipril content at each concentration
level on five consecutive days by initial rate and fixed time
Table 5. Determination of Ramipril in Drug Formulations by Standard Addition Method

Initial rate method
-1
Formulations
Capsule
Hopace-1.25
(Cardicare)
Ramipres-1.25
(Cipla)
Tablet
Ramace-1.25
(AstraZeneca)
Variace-1.25
(Win Medicare)
Concentration (g mL )
Theoretical
Spiked
Nominal SD*
Recovery RSD* (%)
20.0
30.0
20.0
30.0
20.0
40.0
20.0
40.0
39.993
70.044
40.008
69.988
0.078
0.117
0.101
0.117
99.985
100.063
100.021
99.983
20.0
30.0
20.0
30.0
20.0
40.0
20.0
40.0
40.022
70.016
40.008
70.002
0.105
0.131
0.101
0.125
100.056
100.023
100.020
100.003
SAE
CL
0.195
0.167
0.251
0.167
0.034
0.052
0.045
0.052
0.097
0.146
0.125
0.145
0.263
0.187
0.251
0.179
0.047
0.059
0.045
0.056
0.131
0.163
0.125
0.155
SAE
CL
Fixed time method

-1
Formulations
Capsule
Hopace-1.25
(Cardicare)
Ramipres-1.25
(Cipla)
Tablet
Ramace-1.25
(AstraZeneca)
Variace-1.25
(Win Medicare)
Concentration (g mL )
Theoretical
Spiked
Nominal SD*
20.0
30.0
20.0
30.0
20.0
40.0
20.0
40.0
39.993
70.012
40.005
69.999
0.053
0.063
0.050
0.053
99.980
100.017
100.013
99.999
0.133
0.089
0.112
0.075
0.024
0.028
0.020
0.024
0.066
0.078
0.056
0.065
20.0
30.0
20.0
30.0
20.0
40.0
20.0
40.0
39.993
70.037
40.005
69.999
0.053
0.072
0.078
0.083
99.980
100.053
100.013
99.998
0.133
0.103
0.194
0.118
0.024
0.032
0.035
0.037
0.066
0.089
0.096
0.102
E549
Recovery RSD* (%)

Table 6. Point Hypothesis Test: Comparison of the Proposed Methods with the Reference Method at 95% Confidence Level
Initial rate method
Formulations
Capsule
Hopace-1.25
(Cardicare)
Ramipres-1.25
(Cipla)
Tablet
Ramace-1.25
(AstraZeneca)
Variace-1.25
(Win Medicare)
Fixed time method
Reference method
Recovery
%
RSD*
%
t-value
F-value
Recovery
%
RSD*
%
t-value
F-value
Recovery
%
RSD*
%
99.988
0.399
0.123
1.853
100.045
0.188
0.053
2.426
100.070
0.293
100.083
0.337
0.092
1.687
99.961
0.258
0.151
1.018
100.035
0.260
99.988
0.400
0.074
2.359
100.045
0.188
0.023
1.906
100.035
0.260
99.988
0.400
0.074
2.359
99.961
0.258
0.151
1.018
100.035
0.260
Theoretical t-value ( = 8) and F-value ( = 4, 4) at 95% confidence level are 2.306 and 6.39, respectively.
methods (Table 4). The results of standard deviation (SD),

relative standard deviation (RSD) and recoveries by initial
rate and fixed time methods in Table 3 and 4 can be considered to be very satisfactory. Thus the proposed methods are
very effective for the assay of ramipril in drug formulations.
The validity of the proposed methods was presented by recovery studies using the standard addition method. For this
purpose, a known amount of reference drug was spiked to
formulated tablets and capsules at two different concentration levels and the nominal value of drug was estimated
by the proposed method. Each level was repeated five times.
The results (Table 5) were reproducible with low SD and
RSD. No interference from the common excipients was
observed. The applicability of the proposed methods for the
determination of ramipril has been tested on commercially
available pharmaceutical formulations. The results of the
Table 7. Interval Hypothesis Test: Comparison of the Proposed
Methods with the Reference
Formulations
Capsule
Hopace-1.25
(Cardicare)
Ramipres-1.25
(Cipla)
Tablet
Ramace-1.25
(AstraZeneca)
Variace-1.25
(Win Medicare)
Initial rate method
Fixed time method
Lower
limit*
(L)
Upper
limit*
(U)
Lower
limit*
(L)
Upper
limit*
(U)
0.981
1.017
0.987
1.013
0.986
1.015
0.986
1.013
0.982
1.018
0.988
1.012
0.982
1.017
0.986
1.013
proposed method (initial rate or fixed time) were compared

with those obtained by the reference method8 using point
hypothesis test. The students t and F-values (Table 6) at
95% confidence level did not exceed the tabulated t- and
F-values, confirming no significant differences between the
performance of the proposed methods and the reference
method. Once this is done, the evaluation of the bias is made
using the interval hypothesis test as an alternate. Therefore,
the interval hypothesis test25 has been performed to compare
the results of the proposed methods with those of the reference method at 95% confidence level (Table 7). The Canadian Health Protection Branch has recommended that a
bias, based on recovery experiments, of 2% is acceptable.26
It is evident from Table 7 that the true bias of all samples is
smaller than 2%. The interval hypothesis test has also
confirmed that accuracy and precision are within the acceptable limits and indicated no significant differences between the performance of the methods compared at 95%
confidence level.
CONCLUSION
*In pharmaceutical analysis, a bias, based on recovery experiments, of

2% (L = 0.98 and U = 1.02) is acceptable.
The proposed methods are quite simple and do not require

any pretreatment of the drug and tedious extraction procedure. The methods have wider linear dynamic range with
good accuracy and precision. Point and interval hypothesis
tests proved that the proposed methods have acceptable
bias of 2%. Hence the data presented in the manuscript
by kinetic spectrophotometric method for the determination
of ramipril in pharmaceutical formulations demonstrate that
the proposed method is accurate, precise, linear, specific
and robust for the determination of ramipril in tablets and
capsules and thus can be extended for routine analysis of
ramipril in pharmaceutical industries, hospitals and research laboratories.
E550
ACKNOWLEDGMENTS
The authors are grateful to Chairman, Department of Chemistry, Aligarh Muslim University, Aligarh for providing research facilities. Financial assistance provided by Council
of Scientific and Industrial Research (CSIR), New Delhi,
India to Dr. Syed Najmul Hejaz Azmi (snhazmi@yahoo.com)
as Research Associate (Award No. 9/112(329)/2002-EMR-I)
is gratefully acknowledged. The authors wish to express
their gratitude to Dr. Reddys Laboratories Limited (Andhra
Pradesh, India) for the sample of reference standard of pure
ramipril.
11. Blaih SM, Abdine HH, El-Yazbi FA, Shaalan RA. Spectrophotometric determination of enalapril maleate and ramipril in dosage forms.
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Spectrosc Lett. 2001;34:211Y220.
13. Al-Majed AA, Al-Zehouri J. Use of 7-fluoro-4-nitrobenzo-2oxo-1,3-diazole (NBD-F) for the determination of ramipril in tablet and
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15. Sierra-Jimenez F, Sanchez-Pedreno C. The indicator system Hg2+diphenylcarbazide. Anales real Soc Espan Fis y quim. 1958;54B:541Y552.
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AAPS PharmSciTech 2005; 6 (3) Article 68 (http://www.aapspharmscitech.org).